Genetic evidence for causal effects of leukocyte counts on risk for rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by the accumulation of leukocytes and inflammatory mediators within the synovial tissue. Leukocyte counts are proposed to play a role in the pathogenesis of RA. However, the causality remains unclear. To investigate the causal relationship between various leukocytes and RA by implementing two-sample univariable Mendelian Randomization (MR) and multivariable MR. MR analysis was performed using respective genome-wide association study (GWAS) summary statistics for the exposure traits (eosinophil counts, neutrophil counts, lymphocyte counts, monocyte counts, basophil counts, and white blood cell counts) and outcome trait (RA). Summary statistics for leukocytes were extracted from the Blood Cell Consortium meta-analysis and INTERVAL studies. Public GWAS information for RA included 14,361 cases and 43,923 controls. Inverse variance weighted, weighted median, MR-Egger regression, MR pleiotropy residual sum and outlier, and multivariable MR analyses were performed in MR analysis. Univariable MR found elevated eosinophil counts (OR 1.580, 95% CI 1.389–2.681, p = 1.30 × 10–7) significantly increased the risk of RA. Multivariable MR further confirmed that eosinophil counts were a risk factor for RA. Increased eosinophils were associated with higher risk of RA. Further elucidations of the causality and mechanisms underlying are likely to identify feasible interventions to promote RA prevention.


Selection of IVs
IVs extracted meet three criteria to ensure the credibility of this study: SNPs are significantly associated with leukocytes; SNPs have to be independent of confounders affecting leukocytes and the RA; SNPs have to influence the RA through leukocytes rather than through other ways 20 (Fig. 1).
First, genome-wide significant (p < 5 × 10 -8 ) SNPs associated with leukocytes were extracted as the instrumental variables.These SNPs were independent by excluding SNPs in linkage disequilibrium (r 2 < 0.01, clumping window = 10,000 kb).Besides, we queried and removed SNPs associated with confounders in PhenoScanner to exclude potential pleiotropic effects.We also considered and removed palindromic SNPs.Finally, the F statistic was calculated to judge the strength of the genetic instrument variables in the results.An F-statistic ≥ 10 indicates strong relation between the instrumental variables and exposure 21 .

MR analysis
To assure the consistency of data, we harmonized the statistics of leukocytes and RA and subsequently conducted them.Subsequently, we conducted various MR methods to evaluate the causal associations between leukocytes and RA.For the univariate MR analysis, inverse-variance weighted (IVW) was the primary method.The MR-Egger regression and weighted median were also used to evaluate causal relationships.The IVW acquires a robust causal estimate, but it is biased when the SNPs are pleiotropy because of the ignorance of intercept 22 .And the other methods were used for further supplementary analyses.The MR-Egger regression provides estimates after correcting for pleiotropy, although the MR-Egger method is weak in statistical ability 23 .The weighted median also attains robust causal estimates if half of the instrument variables analyzed are invalid 24 .
Besides, we conducted MVMR analysis to research the direct effects of various white blood cell counts (lymphocyte, neutrophil, monocyte, eosinophil, and basophil) on RA.MVMR analysis is an extension of MR analysis that probes the causal effects of multiple factors.It is effective in cases where two or more exposures are related and can help identify whether some directions have a causal impact on the outcome or whether one exposure may be mediated through the effect of the others 25 .

Sensitivity analysis
We conducted various approaches to detect the presence of heterogeneity or pleiotropy.In the study, the p-value of Cochrane's Q was used to assess the degree of heterogeneity, where p > 0.05 suggested no heterogeneity.MR-Egger regression intercept was used to determine the horizontal pleiotropy.The MR pleiotropy residual sum and outlier (MR-PRESSO) method was applied to detect outlier SNPs in IVW linear regression and correct the MR estimation by removing these outliers.Moreover, a leave-one-out analysis was conducted to avoid a single SNP causing horizontal pleiotropy.Finally, funnel, scatter, and leave-one-out plots were also constructed for visual inspection.

Statistical analysis
Bonferroni correction was used for multiple-comparison correction.MR results with p < 8.33 × 10 -3 (0.05/6) and p < 0.05 were considered to have statistically significant for univariable MR and multivariable MR, respectively.The estimated relative risk was the odds ratio and 95% confidence interval (CI).All statistical analyses mentioned above were conducted using R (version 4.2.2), and the "TwoSampleMR" package, the "MVMR" package, and the "MRPROSSO" package were used for MR analysis.

Ethics approval and consent to participate
The summary statistics don't contain any personal information and the original GWAS data of leukocyte counts and RA have obtained ethical approval from relevant ethics review committees.

The character of SNP and participants for analysis
We included genome-wide significant SNPs (p < 5 × 10 -8 ).Then, these SNPs were clustered based on linkage disequilibrium (r 2 < 0.01).The mean F-statistic ranged from 119.57 to 176.34, indicating a low risk of weak instrument bias.At the same time, the harmonized algorithm removes all SNPs that have palindromic structures.In the SNP associated with the leukocytes counts after the screening, 425, 353, 423, 368, 159, and 409 SNPs were included as candidate IVs for MR analysis of total lymphocyte, neutrophil, monocyte, eosinophil, basophil counts and white blood cell counts with RA, respectively (Fig. 2) (Tables S1-S6) 26 .

The causal effect of eosinophil counts on RA in validation stage
In the validation stage, we successfully replicated the MR results of eosinophil counts on RA.Univariable MR results demonstrated that eosinophil counts had a significant causal effect on RA (OR 1.206, 95% CI 1.040-1.398,p = 0.013).The scatter plot and plot of leave-one-out analyses were displayed in Fig. S1.The information of IVs of eosinophil counts is shown in Table S7.

Sensitivity analysis validation
In this study, a variety of sensitivity analysis methods were used.First, Cochran's Q-test assessed heterogeneity between individual SNP estimates.For heterogeneity, it was observed in all outcomes: neutrophil counts (p = 0), lymphocyte counts (p = 9.69 × 10 -274 ), monocyte counts (p = 9.50 × 10 -44 ), eosinophil counts (p = 0) and basophil counts (p = 1.94 × 10 -51 ), and white blood cell counts (p = 4.74 × 10 -280 ).Therefore, we choose the random effects model to evaluate the causal effect 27 .Second, we used the MR-Egger intercept method to test the horizontal pleiotropy of IVs.There was no horizontal pleiotropy (p > 0.05) for each result.Then, Map funnels and forests show no presence of pleiotropy (Figs.S2, S3) 28 .At last, in the "leave-one-out" analysis, eosinophil counts were to the right of the 0 vertical lines (Fig. 4), indicating the classification result was deterministic.

Discussion
In this study, we performed a univariate versus multivariate analysis to assess the causal relationship between types of blood leukocyte counts and RA.Univariate MR analysis revealed that eosinophils had a causal relationship with RA.The causal relationship between eosinophils and RA was more robust after correcting the interaction between different leukocyte traits 16 .The increase in eosinophils predicts higher disease activity in RA, consistent with previous studies 29,30 .Eosinophils regulate adaptive immune responses and play an essential role in inflammatory and autoimmune diseases 31 .Observational studies have documented persistent increased eosinophilia in patients with RA 11,32 .The survey by Dewi Guellec et al. 8 found that patients with mild eosinophilia were less responsive to treatment.The role of eosinophils on risk factors may be related to the eosinophil cationic protein (ECP), mediators stored in granules throughout the cytoplasm synthesized by eosinophils 33 .ECP, released from the activated eosinophils, is a ribonuclease with neurotoxic, cytotoxic, fibrosis-promoting, and immunomodulatory functions.Eosinophils release ECP in two ways: antibody-dependent (immunoglobulin) and antibody-independent (C3 and C5 complement components) activation of eosinophil degranulation 34,35 .Signaling by ECP induces the release of several proinflammatory factors/chemokines, including C30L and S100 proteins 36 , and may cause inflammation in a variety of tissue types 36 .The upregulation of CD30L is considered involved in RA pathogenesis 37 .We speculate that this is a possible mechanism of CD30L involvement in the immune process of RA.Moreover, previous studies have demonstrated that CD30 values are higher in active RA patients than in inactive RA and are directly related to rheumatoid factor serum titer 38,39 .
Due to the dual nature of eosinophils as pro-inflammatory and pro-resolving cells 40 , the association of eosinophils with the development of RA is still controversial.Darja Andreev et al. 12 found that eosinophils' proresolving properties in RA.Eosinophils promote the resolution of inflammation by producing pro-resolving lipid mediators through the 1/12-LOX-mediated biosynthetic pathway 41 .It was also demonstrated that eosinophils exert anti-inflammatory effects in arthritis by inducing M38 macrophage polarization by inhibiting the IκB/P2 MAPK signaling pathway 42 .Based on the different effects of eosinophils on RA, it can be speculated that eosinophils have subpopulations responsible for various biological functions.This conjecture has been confirmed that a population of regulatory eosinophils (rEos) exists in the joint that promotes the production of alternatively activated macrophages by generating IL-4, IL-13, and 12 / 15-lox-derived media 12 .Our MR analysis found that genetically predicted increased eosinophils were associated with an increased risk of RA, suggesting that the pro-inflammatory effect of eosinophils may be the primary driver of the increased risk of RA.
Moreover, our MR analysis did not indicate a causal relationship between other cell counts and RA except eosinophils.Although experimental studies have found that the absolute number of basophils increases in children with RA 43 , the causality remains unclear.Our univariate MR analysis confirms several observational studies, but after correction by multivariate MR analysis, this potential causality is no longer reliable.Previous studies may be limited by confounding factors and reverse causality.Studies have demonstrated an association between peripheral blood neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), and RA 44,45 , but our study did not find a causal relationship between neutrophil counts, lymphocyte counts, monocyte counts, and RA, indicating that NLR and LMR may be the main factor reflecting the potential pathogenesis of RA and disease progression.More studies are needed to confirm this.
Our study used the MR method to mitigate confounding bias and removed confounding factors by PhenoScanner.The robustness of the results was ensured by using three MR analysis techniques and a detailed sensitivity analysis.The large sample size from the white blood cell counts provided the strength of the tool variables (F statistic > 10).A limitation of this study is the need for detailed phenotypic data in available GWAS, which prevented us from testing the association of specific eosinophilic subtypes with RA, such as the risk of RA with rEOS counts as an exposure factor.In the future, more comprehensive GWAS data may allow stratified analyses of different cell subtypes.

Conclusions
The current study remains controversial regarding the association of leukocytes with RA.Our MR study found that a higher genetically predicted risk of RA was associated with increased eosinophils but not with neutrophils, basophils, monocytes, and neutrophils.The association of RA with eosinophils may differ by different cell subsets, and more studies are needed to explore eosinophils' effects on the pathogenesis of RA.

Figure 3 .
Figure 3. Multivariable MR analysis forest plot: effect of multiple risk factors on RA.OR, odds ratio; CI, confidence interval.

Figure 4 .
Figure 4. Sensitivity analysis of causal effect between eosinophil counts and RA based on the leave-one-out approach.

Table 1 .
Univariable MR estimates of types of blood leukocyte counts on the risk of RA.OR odds ratio, 95% CI 95% confidence interval.Exposure MR methodology N